The conformational dynamics of transcriptional activators, coactivators and their complexes underpin regulated transcription. The low energy barriers between individual conformations mean that each participant can use the same group of amino acids to recognize a variety of binding partners, with each complex assuming a distinct conformation. The assumption is that each of the conformations correlates with a unique functional outcome via allosteric communication with other binding partners. However, this model has never been tested, primarily due to a lack of robust tools to connect in vitro observations with function in cells. Our goal is to develop and implement chemical genetic tools for this purpose. Focusing on two functionally important coactivator motifs, KIX (neuropathic pain, neurodegenerative disorders) and AcID (cancer, viral infection), we will develop covalent chemical co-chaperones that stabilize particular conformations of the coactivators alone and in complex with cognate ligands. These co-chaperones will be used to rigorously characterize the structure (X-ray crystallography, NMR spectroscopy) and dynamics (transient kinetics, equilibrium binding, computational analysis) of the coactivators in distinct conformational and assembly states. Complementing the in vitro data will be experiments with chemical co-chaperones in cells, thus identifying both the interactions and the conformations that regulate transcriptional output. Through these studies, allosteric binding sites for small molecule targeting of these disease-relevant transcriptional complexes will be identified.